Abstract

The investigation of vertebrate limb regeneration, a favorite topic of early developmental
biologists, is enjoying a renaissance thanks to recently developed molecular and genetic
tools, as indicated in recent papers in BMC Biology and BMC Developmental Biology. Classical experiments provide a rich context for interpreting modern functional
studies.

Minireview

Nearly 240 years have passed since the first scientific treatise addressing limb regeneration,
Spallanzani's 'Reproduction of the Legs in the Aquatic Salamander' within his An Essay on Animal Reproductions [1]. In spite of extraordinary advances in other areas of developmental biology in the
past few decades, many of the most remarkable features of limb regeneration outlined
by Spallanzani remain mysterious today. However, recent advances in genomics and molecular
biology offer the potential to finally illuminate the cellular and molecular mechanisms
underlying amphibian limb regeneration. Changes in gene expression accompanying regenerative
events can now be profiled by microarrays. Recent projects by Monaghan et al. [2] published in BMC Biology and by Pearl et al. [3] in BMC Developmental Biology have provided thousands of cDNA sequences of transcripts expressed during limb regeneration
in amphibians. Moreover, the newly developed application of transgenesis to axolotl
salamanders [4] suggests that functional roles for specific genes are likely to be elucidated in
the near future. As these tools are brought to bear on the problem of limb regeneration,
work will build on and be guided by the extensive classical literature, including
both experimental and descriptive studies.

Wound healing makes all the difference

Following amputation, a salamander's limb bleeds only briefly and the important operation
of healing the wound in a way conducive to regeneration begins. Within 24 hours, the
cut surface is ensheathed by epithelial cells that migrate from the surface of the
stump (Figure 1). These 'wound epidermis' cells proliferate, forming the 'apical epidermal cap' (AEC),
a structure postulated to provide key molecular signals needed to stimulate and/or
maintain the early stages of regeneration. Without this specialized wound healing,
regeneration fails; for instance, if the limb is amputated and the dorsal and ventral
skin is pulled together and sutured, no true AEC forms and the limb remains a stump.

Figure 1.Key morphological events of vertebrate limb regeneration. Following amputation, epidermal cells from the surface of the stump rapidly migrate
to cover the wound (1), forming the apical epidermal cap (AEC, red). Stump cells are
used to create a blastema (blue) beneath the AEC (2). Blastema cells proliferate and
the structure acquires a cone-shaped morphology (3). Undifferentiated blastema cells
begin to differentiate into various cell-types within the newly formed limb (4). The
new portion continues to grow. Once patterning and growth are complete, a perfectly
functional new limb has been regenerated (5).

Building a blastema

The next critical step is to create a blastema – a pool of cells from which the new
limb will arise. Forming at the distal tip of the old stump but beneath the AEC, the
blastema morphologically appears as a transparent outgrowth that acquires the shape
of a cone as regeneration proceeds (Figure 1). Blastema cells are thought to be relatively undifferentiated mesenchymal cells,
but their origins remain highly controversial (reviewed in [5]). Early work suggested that at least some blastema cells arise by the dedifferentiation
of muscle fibers, as the fibers immediately adjacent to the amputation plane showed
microscopic signs of cellularization, and these presumably newly created mononucleate
cells incorporated tritiated thymidine [6]. Studies using modern labeling techniques, such as fluorescent dye tracking and fluorescently
labeled antibodies, support a similar model, yet controversy remains because others
claim that a stem-cell population, the muscle satellite cells, also participate in
blastema formation. Furthermore, the possibility of transdifferentiation of cells
in the stump to different cell types in the regenerate, a process hinted at in earlier
studies, needs to be definitively addressed, both in terms of the potential of blastema
cells for transdifferentiation and the extent to which this phenomenon is significant
for normal regeneration. These questions await more sophisticated cell-lineage analysis.
Such analysis may be facilitated by the identification of cell-type-specific promoters
in conjunction with the recently developed transgenic approaches.

Once the blastema cells are collected under the AEC, they must proliferate to provide
enough cells to drive the regeneration process forward (Figure 1). The proliferation of blastema cells has been shown to be critically reliant on
the presence of the nerve in the limb [7]. For example, a limb that has been denervated and then amputated will close the wound
in an outwardly normal manner, and a blastema will form, but the blastema cells do
not proliferate enough and regeneration fails. Interestingly, if a limb is manipulated
to develop originally without the nerve, this limb can be amputated and a fairly normally
regenerated limb grows. These data suggest the limb somehow becomes 'addicted' to
factors produced by the nerve and then needs them for regeneration.

Recent work has shown that regeneration of a denervated limb can be mostly rescued
by providing cDNA encoding a single protein, nAG [8]. nAG is a secreted ligand for Prod1, a hitherto mysterious cell-surface molecule
whose expression is graded along the proximal-distal axis in a salamander limb. A
yeast two-hybrid strategy was used to uncover nAG, and the relatively modern technique
of electroporation of plasmid DNA into limb blastemas was used to demonstrate its
sufficiency for replacing the nerve.

While the outlines of blastema formation are fairly well understood, relatively few
molecules have been implicated in specific events that form and shape the blastema.
Much work remains to discover the cellular origins of blastema cells, how these cells
are cued to form a blastema, and how the blastema cells are stimulated to proliferate.
Some clues may be found using genomic approaches, as shown by the recent study by
Monaghan et al. [2], where many transcripts were identified as differentially expressed in blastemas
undergoing normal regeneration compared with those whose limb had been denervated.

Finishing the job

Eventually, blastema cells begin the process of reorganizing and of specifying distinct
cellular identities for the new limb. Morphologically, the blastema becomes flattened
and acquires the shape characteristic of a 'palette-staged' limb bud with the vague
outline of future digits discernable (Figure 1). Most of the events governing the regeneration process from this point onward are
presumed to be similar or identical to the molecular events that transform a limb
bud into a limb. It is, however, important to note that many of these assumptions
remain to be tested, and that the two scenarios cannot be completely equivalent. For
instance, the scale at which a limb regenerates is often many times – perhaps even
thousands of times – larger than that at which it developed when the animal was a
tiny larva. In addition, new features such as blood vessels and fine nerves need to
be seamlessly integrated into the existing structures on the stump if the limb is
to thrive and function properly. Nonetheless, some mechanisms have already been shown
to be common; for example, the ectopic production of Sonic hedgehog signaling activity
in the anterior margin of a regenerating limb produces the same effect – duplication
of posterior digits – in a regenerating blastema as in a newly developing limb bud
[9].

Decoding the secrets of perfect regeneration

If all steps proceed normally, the salamander or tadpole regrows a perfect replica
of its original limb. This precise replication is one of the most remarkable aspects
of regeneration. An animal that loses a foot will grow back only a foot and no more;
one that loses the leg from the thigh will grow back everything that was once distal
to the thigh's amputation plane. Somehow, the salamander's body can measure where
the amputation occurred along the proximal-distal axis and replace only the missing
part, but how?

While the process is still poorly understood, some clues have come from blastema-grafting
experiments (reviewed in [10]). When grafted to a proximal 'thigh' blastema, a distal blastema 'fated' to make
a foot translocates distally with the host's regenerating limb and gives rise to a
regenerated limb that essentially has two feet. Alternatively, a proximal blastema
grafted to a proximal blastema host will create a salamander with essentially two
complete legs. Therefore, the proximo-distal information is encoded within the blastema.
Remarkably, if a proximal limb blastema is grafted to a receptive field such as the
eye (parts of which can also regenerate in many salamanders), a limb will grow from
the eye socket, demonstrating that the blastema is indeed an autonomous unit and,
once created, may only rely on the underlying tissue for survival factors but not
for contextual information. On a molecular level, there is evidence that the cell
surface protein Prod1, mentioned above, plays a critical role in mediating proximo-distal
positional information. However, the question of how positional information is established
in the blastema and how it influences cell behavior to achieve precise replacement
of amputated structures remains largely untouched but will benefit from the application
of the modern genomic and genetic techniques discussed earlier.

Understanding the molecular and cellular mechanisms that allow salamanders to create
and develop a blastema may help develop therapies for improving regeneration in animals
that do not. A good starting point for comparison is a salamander, which can regenerate
throughout its life, and a frog, which can only regenerate limbs while it is a tadpole
and gradually loses the ability to regenerate as it approaches the final step of metamorphosis.
An even simpler comparison can be made between a tadpole at a stage that regenerates
versus a later-staged tadpole that cannot. The recent work from Caroline Beck's lab
(Pearl et al. [3]) profiled gene expression in blastemas from normally regenerating tadpoles compared
with those in which regeneration was blocked by the misexpression of Noggin, an inhibitor
of the secreted signal molecule bone morphogenetic protein (BMP). Genes defined as
essential regulators of regeneration in this case included those that specifically
influence the transition from an early blastema to a larger, cone-shaped blastema
(the step that is blocked in the absence of BMP activity).

Similar approaches may prove fruitful for discovering transcripts expressed at other
discrete stages, for instance, during the critical wound healing that initiates limb
regeneration in the salamander. Further evidence for the importance of this step comes
from human medicine: in young children with distal amputations of digits, regeneration
of a perfect fingertip can occur, but only if the stump skin is not sutured together.
If early healing stages were better understood in both regenerating and non-regenerating
scenarios, we would have a better chance of figuring out how to heal a wound in a
way that leads to formation of a blastema.

Regeneration research is now undergoing a resurgence, with initial efforts fueled
by modern approaches to understanding gene expression. Upcoming work will take advantage
of the power of transgenesis to explicitly address the functions of specific genes
at particular stages of regeneration and in particular cell types. Additional tools
are still needed, however. Limb regeneration is most impressive among salamanders,
and no salamander genomes have been sequenced to date (mostly due to their enormous
size). Moreover, a reliable method for eliminating or reducing gene function in salamanders
has not yet been established. As such new genetic and genomic tools are developed,
we will be able to fully realize the power of salamanders as model systems for understanding
limb regeneration.

Acknowledgements

Regeneration research in the Tabin lab is funded by the NIH, grants R01 HD045499,
1F32 HD054082-01.

References

Spallanzani A: Reproductions of the legs in the aquatic salamander. In An Essay on Animal Reproductions. London: Becket and de Hondt; 1769:68-82.

Sobkow L, Epperlein HH, Herklotz S, Straube WL, Tanaka EM: A germline GFP transgenic axolotl and its use to track cell fate: dual origin of the
fin mesenchyme during development and the fate of blood cells during regeneration.